Nucleic acid encoding GAI gene of Arabidopsis thaliana

ABSTRACT

This invention relates to the genetic control of growth and/or development of plants and the cloning and expression of genes involved therein. More particularly, the invention relates to the cloning and expression of the GAI gene of  Arabidopsis thaliana,  and use of the gene in plants.

This invention relates to the genetic control of growth and/ordevelopment of plants and the cloning and expression of genes involvedtherein. More particularly, the invention relates to the cloning andexpression of the GAI gene of Arabidopsis thaliana, and homologues fromother species, and use of the genes in plants.

BACKGROUND OF THE INVENTION

An understanding of the genetic mechanisms which influence growth anddevelopment of plants, including flowering, provides a means foraltering the characteristics of a target plant. Species for whichmanipulation of growth and/or development characteristics may beadvantageous includes all crops, with important examples being thecereals, rice and maize, probably the most agronomically important inwarmer climatic zones, and wheat, barley, oats and rye in more temperateclimates. Important crops for seed products are oil seed rape andcanola, sugar beet, maize, sunflower, soyabean and sorghum. Many cropswhich are harvested for their roots are, of course, grown annually fromseed and the production of seed of any kind is very dependent upon theability of the plant to flower, to be pollinated and to set seed. Inhorticulture, control of the timing of growth and development, includingflowering, is important. Horticultural plants whose flowering may becontrolled include lettuce, endive and vegetable brassicas includingcabbage, broccoli and cauliflower, and carnations and geraniums. Dwarfplants on the one hand and over-size, taller plants on the other may beadvantageous and/or desirable in various horticultural and agriculturalcontexts.

Arabidopsis thaliana is a favourite of plant geneticists as a modelorganism. Because it has a small, well-characterized genome, isrelatively easily transformed and regenerated and has a rapid growingcycle, Arabidopsis is an ideal model plant in which to study growth anddevelopment and its control.

Many plant growth and developmental processes are regulated by specificmembers of a family of tetracyclic diterpenoid growth factors known asgibberellins (GA)¹. The gai mutation of Arabidopsis confers a dwarfphenotype and a dramatic reduction in GA-responsiveness²⁻⁹. Here wereport the molecular cloning of gal via Ds transposon mutagenesis.

The phenotype conferred by the Ds insertion allele confirms that gai isa gain-of-function mutation, and that the wild-type allele (GAI) isdispensable^(5,6). GAI encodes a novel polypeptide (GAI) of 532 aminoacid residues, of which a 17 amino acid domain is missing in the gaimutant polypeptide. This result is consistent with GAI acting as a plantgrowth repressor whose activity is antagonized by GA. Though we are notto be bound by any particular theory, gai may repress growthconstitutively because it lacks the domain that interacts with the GAsignal. Thus according to this model GA regulates plant growth byde-repression.

gai is a dominant, gain-of-function mutation, which confers adark-green, dwarf phenotype, and interferes with GA reception orsubsequent signal-transduction²⁻⁹. Dominant mutations conferring similarphenotypes are known in other plant species, including maize¹⁰⁻¹² andwheat¹³. The latter are especially important because they are the basisof the high-yielding, semi-dwarf wheat varieties of the ‘greenrevolution’¹⁴. The increased yield of these varieties is due to anincreased grain production per ear, and superior straw strength. Theshorter, stronger straw greatly reduces the losses resulting fromlodging, that is flattening of standing wheat plants by rain/wind. Weset out to clone gal from Arabidopsis because of its importance to theunderstanding of GA signal-transduction, and because of the potentialfor use of GA-insensitivity in the development of wheat and other cropssuch as oil-seed rape and rice which may show improvement as great asthat already seen in wheat.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention there is provided anucleic acid molecule comprising a nucleotide sequence encoding apolypeptide with GAI function. The term “GAI function” indicates abilityto influence the phenotype of a plant like the GAI gene of Arabidopsisthaliana. “GAI function” may be observed phenotypically in a plant asinhibition, suppression, repression or reduction of plant growth whichinhibition, suppression, repression or reduction is antagonised by GA.GAI expression tends to confer a dwarf phenotype on a plant which isantagonised by GA. Overexpression in a plant from a nucleotide sequenceencoding a polypeptide with GAI function may be used to confer a dwarfphenotype on a plant which is correctable by treatment with GA.

Also according to an aspect of the present invention there is provided anucleic acid molecule comprising a nucleotide sequence encoding apolypeptide with ability to confer a gai mutant phenotype uponexpression. gai mutant plants are dwarfed compared with wild-type, thedwarfing being GA-insensitive.

By gibberellin or GA is meant a diterpenoid molecule with the basiccarbon-ring structure shown in FIG. 1 and possessing biologicalactivity, i.e. we refer to biologically active gibberellins.

Biological activity may be defined by one or more of stimulation of cellelongation, leaf senescence or elicitation of the cereal aleuroneα-amylase response. There are many standard assays available in the art,a positive result in any one or more of which signals a test gibberellinas biologically active^(28,29,30).

Assays available in the art include the lettuce hypocotyl assay,cucumber hypocotyl assay, and oat first leaf assay, all of whichdetermine biological activity on the basis of ability of an appliedgibberellin to cause elongation of the respective tissue. Preferredassays are those in which the test composition is applied to agibberellin-deficient plant. Such preferred assays include treatment ofdwarf GA-deficient Arabidopsis to determine growth, the dwarf pea assay,in which internode elongation is determined, the Tan-ginbozu dwarf riceassay, in which elongation of leaf sheath is determined, and thed5-maize assay, also in which elongation of leaf sheath is determined.The elongation-bioassays measure the effects of general cell elongationin the respective organs and are not restricted to particular celltypes.

Further available assays include the dock (Rumex) leaf senescence assayand the cereal aleurone α-amylase assay. Aleurone cells which surroundthe endosperm in grain secrete α-amylase on germination, which digestsstarch to produce sugars then used by the growing plant. The enzymeproduction is controlled by GA. Isolated aleurone cells givenbiologically active GA secrete α-amylase whose activity can then beassayed, for example by measurement of degradation of starch.

Structural features important for high biological activity (exhibited byGA₁, GA₂, GA₄ and GA₇) are a carboxyl group on C-6 of B-ring; C-19, C-10lactone; and β-hydroxylation at C-3. β-hydroxylation at C-2 causesinactivity (exhibited by GA₈, GA₂₉, GA₃₄ and GA₅₁). gai mutants do notrespond to GA treatment, e.g. treatment with GA₁, GA₃ or GA₄.

Treatment with GA is preferably by spraying with aqueous solution, forexample spraying with 10⁻⁴M GA₃ or GA₄ in aqueous solution, perhapsweekly or more frequently, and may be by placing droplets on plantsrather than spraying. GA may be applied dissolved in an organic solventsuch as ethanol or acetone, because it is more soluble in these than inwater, but this is not preferred because these solvents have a tendencyto damage plants. If an organic solvent is to be used, suitableformulations include 24 ηl of 0.6, 4.0 or 300 mM GA₃ or GA₄ dissolved in80% ethanol. Plants, e.g. Arabidopsis, may be grown on a mediumcontaining GA, such as tissue culture medium (GM) solidified with agarand containing supplementary GA.

Nucleic acid according to the present invention may have the sequence ofa wild-type GAI gene of Arabidopsis thaliana, or be a mutant,derivative, variant or allele of the sequence provided. Preferredmutants, derivatives, variants and alleles are those which encode aprotein which retains a functional characteristic of the protein encodedby the wild-type gene, especially the ability for plant growthinhibition, which inhibition is antagonised by GA, or ability to conferon a plant one or more other characteristics responsive to GA treatmentof the plant. Other preferred mutants, derivatives, variants and allelesencode a protein which confers a gai mutant phenotype, that is to sayreduced plant growth which reduction is insensitive to GA, i.e. notovercome by GA treatment. Changes to a sequence, to produce a mutant,variant or derivative, may be by one or more of addition, insertion,deletion or substitution of one or more nucleotides in the nucleic acid,leading to the addition, insertion, deletion or substitution of one ormore amino acids in the encoded polypeptide. Of course, changes to thenucleic acid which make no difference to the encoded amino acid sequenceare included.

A preferred nucleotide sequence for a GAI gene is one which encodesamino acid sequence shown in FIG. 4 (SEQ ID NO:2), especially a codingsequence shown in FIG. 3 (SEQ ID NO:1). A preferred gai mutant lackspart or all of the 17 amino acid sequence underlined in FIG. 4.

The present invention also provides a nucleic acid construct or vectorwhich comprises nucleic acid with any one of the provided sequences,preferably a construct or vector from which polypeptide encoded by thenucleic acid sequence can be expressed. The construct or vector ispreferably suitable for transformation into a plant cell. The inventionfurther encompasses a host cell transformed with such a construct orvector, especially a plant cell. Thus, a host cell, such as a plantcell, comprising nucleic acid according to the present invention isprovided. Within the cell, the nucleic acid may be incorporated withinthe chromosome. There may be more than one heterologous nucleotidesequence per haploid genome. This, for example, enables increasedexpression of the gene product compared with endogenous levels, asdiscussed below.

A construct or vector comprising nucleic acid according to the presentinvention need not include a promoter or other regulatory sequence,particularly if the vector is to be used to introduce the nucleic acidinto cells for recombination into the genome. However, in one aspect thepresent invention provides a nucleic acid construct comprising a GAI orgai coding sequence (which includes homologues from other thanArabidopsis thaliana) joined to a regulatory sequence for control ofexpression, the regulatory sequence being other than that naturallyfused to the coding sequence and preferably of or derived from anothergene.

Nucleic acid molecules and vectors according to the present inventionmay be as an isolate, provided isolated from their natural environment,in substantially pure or homogeneous form, or free or substantially freeof nucleic acid or genes of the species of interest or origin other thanthe sequence encoding a polypeptide able to influence growth and/ordevelopment, which may include flowering, eg in Arabidopsis thaliananucleic acid other than the GAI coding sequence. The term “nucleic acidisolate” encompasses wholly or partially synthetic nucleic acid.

Nucleic acid may of course be double- or single-stranded, cDNA orgenomic DNA, RNA, wholly or partially synthetic, as appropriate. Ofcourse, where nucleic acid according to the invention includes RNA,reference to the sequence shown should be construed as reference to theRNA equivalent, with U substituted for T.

The present invention also encompasses the expression product of any ofthe nucleic acid sequences disclosed and methods of making theexpression product by expression from encoding nucleic acid thereforunder suitable conditions in suitable host cells. Those skilled in theart are well able to construct vectors and design protocols forexpression and recovery of products of recombinant gene expression.Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorfragments, polyadenylation sequences, enhancer sequences, marker genesand other sequences as appropriate. For further details see, forexample, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrooket al, 1989, Cold Spring Harbor Laboratory Press. Transformationprocedures depend on the host used, but are well known. Many knowntechniques and protocols for manipulation of nucleic acid, for examplein preparation of nucleic acid constructs, mutagenesis, sequencing,introduction of DNA into cells and gene expression, and analysis ofproteins, are described in detail in Protocols in Molecular Biology,Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992. Specificprocedures and vectors previously used with wide success upon plants aredescribed by Bevan, Nucl. Acids Res. (1984) 12, 8711-8721), andGuerineau and Mullineaux, (1993) Plant transformation and expressionvectors. In: Plant Molecular Biology Labfax (Croy RRD ed) Oxford, BIOSScientific Publishers, pp 121-148. The disclosures of Sambrook et al.and Ausubel et al. and all other documents mentioned herein areincorporated herein by reference.

Since the GAI amino acid sequence of Arabidopsis shown in FIG. 4 (SEQ IDNO:2) includes 5 consecutive histidines close to its N-terminus,substantial purification of GAI or gai may be achieved using Ni-NTAresin available from QIAGEN Inc. (USA) and DIAGEN GmbH (Germany). SeeJanknecht et al³¹ and EP-A-0253303 and EP-A-0282042. Ni-NTA resin hashigh affinity for proteins wiht consecutive histidines close to the N-or C-terminus of the protein and so may be used to purifiy GAI or gaiproteins from plants, plant parts or extracts or from recombinantorganisms such as yeast or bacteria, e.g. E. coli, expressing theprotein.

Purified GAI protein, e.g. produced recombinantly by expression fromencoding nucleic acid therefor, may be used to raise antibodiesemploying techniques which are standard in the art. Antibodies andpolypeptides comprising antigen-binding fragments of antibodies may beused in identifying homologues from other species as discussed furtherbelow.

Methods of producing antibodies include immunising a mammal (eg human,mouse, rat, rabbit, horse, goat, sheep or monkey) with the protein or afragment thereof. Antibodies may be obtained from immunised animalsusing any of a variety of techniques known in the art, and might bescreened, preferably using binding of antibody to antigen of interest.For instance, Western blotting techniques or immunoprecipitation may beused (Armitage et al, 1992, Nature 357: 80-82). Antibodies may bepolyclonal or monoclonal.

As an alternative or supplement to immunising a mammal, antibodies withappropriate binding specificty may be obtained from a recombinantlyproduced library of expressed immunoglobulin variable domains, eg usinglambda bacteriophage or filamentous bacteriophage which displayfunctional immunoglobulin binding domains on their surfaces; forinstance see WO92/01047.

Antibodies raised to a GAI, or gai, polypeptide can be used in theidentification and/or isolation of homologous polypeptides, and then theencoding genes. Thus, the present invention provides a method ofidentifying or isolating a polypeptide with GAI function or ability toconfer a gai mutant phenotype, comprising screening candidatepolypeptides with a polypeptide comprising the antigen-binding domain ofan antibody (for example whole antibody or a fragment thereof) which isable to bind an Arabidopsis GAI or gai polypeptide, or preferably hasbinding specificity for such a polypeptide, such as having the aminoacid sequence shown in FIG. 4 (SEQ ID NO:2).

Candidate polypeptides for screening may for instance be the products ofan expression library created using nucleic acid derived from an plantof interest, or may be the product of a purification process from anatural source.

A polypeptide found to bind the antibody may be isolated and then may besubject to amino acid sequencing. Any suitable technique may be used tosequence the polypeptide either wholly or partially (for instance afragment of the polypeptide may be sequenced). Amino acid sequenceinformation may be used in obtaining nucleic acid encoding thepolypeptide, for instance by designing one or more oligonucleotides(e.g. a degenerate pool of oligonucleotides) for use as probes orprimers in hybridisation to candidate nucleic acid, as discussed furtherbelow.

A further aspect of the present invention provides a method ofidentifying and cloning GAI homologues from plant species other thanArabidopsis thaliana which method employs a nucleotide sequence derivedfrom that shown in FIG. 3 (SEQ ID NO:1). Sequences derived from thesemay themselves be used in identifying and in cloning other sequences.The nucleotide sequence information provided herein, or any partthereof, may be used in a data-base search to find homologous sequences,expression products of which can be tested for GAI function.Alternatively, nucleic acid libraries may be screened using techniqueswell known to those skilled in the art and homologous sequences therebyidentified then tested.

For instance, the present invention also provides a method ofidentifying and/or isolating a GAI or gai homologue gene, comprisingprobing candidate (or “target”) nucleic acid with nucleic acid whichencodes a polypeptide with GAI function or a fragment or mutant,derivative or allele thereof. The candidate nucleic acid (which may be,for instance, cDNA or genomic DNA) may be derived from any cell ororganism which may contain or is suspected of containing nucleic acidencoding such a homologue.

In a preferred embodiment of this aspect of the present invention, thenucleic acid used for probing of candidate nucleic acid encodes an aminoacid sequence shown in FIG. 4 (SEQ ID NO:2), a sequence complementary toa coding sequence, or a fragment of any of these, most preferablycomprising a nucleotide sequence shown in FIG. 3 (SEQ ID NO:1).

Alternatively, as discussed, a probe may be designed using amino acidsequence information obtained by sequencing a polypeptide identified asbeing able to be bound by an antigen-binding domain of an antibody whichis able to bind a GAI or gai polypeptide such as one with the amino acidsequence shown in FIG. 4 (SEQ ID NO:2).

Preferred conditions for probing are those which are stringent enoughfor there to be a simple pattern with a small number of hybridizationsidentified as positive which can be investigated further. It is wellknown in the art to increase stringency of hybridisation gradually untilonly a few positive clones remain.

As an alternative to probing, though still employing nucleic acidhybridisation, oligonucleotides designed to amplify DNA sequences fromGAI genes may be used in PCR or other methods involving amplification ofnucleic acid, using routine procedures. See for instance “PCR protocols;A Guide to Methods and Applications”, Eds. Innis et al, 1990, AcademicPress, New York.

Preferred amino acid sequences suitable for use in the design of probesor PCR primers are sequences conserved (completely, substantially orpartly) between GAI genes.

On the basis of amino acid sequence information, oligonucleotide probesor primers may be designed, taking into account the degeneracy of thegenetic code, and, where appropriate, codon usage of the organism fromthe candidate nucleic acid is derived.

The present invention also extends to nucleic acid encoding a GAIhomologue obtained using a nucleotide sequence derived from that shownin FIG. 3 (SEQ ID NO:1).

Also included within the scope of the present invention are nucleic acidmolecules which encode amino acid sequences which are homologues of thepolypeptide encoded by GAI of Arabidopsis thaliana. A homologue may befrom a species other than Arabidopsis thaliana.

Homology may be at the nucleotide sequence and/or amino acid sequencelevel. Preferably, the nucleic acid and/or amino acid sequence shareshomology with the sequence encoded by the nucleotide sequence of FIG. 3(SEQ ID NO:1), preferably at least about 50%, or 60%, or 70%, or 80%homology, most preferably at least 90% or 95% homology. Nuleic acidencoding such a polypeptide may preferably share with the Arabidopsisthaliana GAI gene the ability to confer a particular phenotype onexpression in a plant, preferably a phenotype which is GA responsive(i.e. there is a change in a characteristic of the plant on treatmentwith GA), such as the ability to inhibit plant growth where theinhibition is antagonised by GA. As noted, GAI expression in a plant mayaffect one or more other characteristics of the plant. A preferredcharacteristic that may be shared with the Arabidopsis thaliana GAI geneis the ability to complement a GAI null mutant phenotype in a plant suchas Arabidopsis thaliana, such phenotype being resistance to the dwarfingeffect of paclobutrazol.

Some preferred embodiments of polypeptides according to the presentinvention (encoded by nucleic acid embodiments according to the presentinvention) include the 17 amino acid sequence which is underlined inFIG. 4 or a contiguous sequence of amino acids residues with at leastabout 10 residues with similarity or identity with the respectivecorresponding residue (in terms of position) in 17 amino acids which areunderlined in FIG. 4, more preferably, 11, 12, 13, 14, 15, 16 or 17 suchresidues.

As is well-understood, homology at the amino acid level is generally interms of amino acid similarity or identity. Similarity allows for“conservative variation”, i.e. substitution of one hydrophobic residuesuch as isoleucine, valine, leucine or methionine for another, or thesubstitution of one polar residue for another, such as arginine forlysine, glutamic for aspartic acid, or glutamine for asparagine.Similarity may be as defined and determined by the TBLASTN program, ofAltschul et al. (1990) J. Mol. Biol. 215: 403-10, which is in standarduse in the art. Homology may be over the full-length of the GAI sequenceof FIG. 4 (SEQ ID NO:2), or may more preferably be over a contiguoussequence of 17 amino acids, compared with the 17 amino acids underlinedin FIG. 4, or a longer sequence, e.g. about 20, 25, 30, 40, 50 or moreamino acids, compared with the amino acid sequence of FIG. 4 (SEQ IDNO:2) and preferably including the underlined 17 amino acids.

At the nucleic acid level, homology may be over the full-length or morepreferably by comparison with the 51 nucleotide coding sequence withinthe sequence of FIG. 3 (SEQ ID NO:1) and encoding the 17 amino acidsequence underlined in FIG. 4, or a longer sequence, e.g. about, 60, 70,80, 90, 100, 120, 150 or more nucleotides and preferably includeing the51 nucleotide of FIG. 3 (SEQ ID NO:1) which encodes the underlined 17amino acid sequence of FIG. 4.

Homologues to gai mutants are also provided by the present invention.These may be mutants where the wild-type includes the 17 amino acidsunderlined in FIG. 4, or a contiguous sequence of 17 amino acids with atleast about 10 (more preferably, 11, 12, 13, 14, 15, 16 or 17) whichhave similarity or identity with the corresponding residue in the 17amino acid sequence underlined in FIG. 4, but the mutant does not.Nucleic acid encoding such mutant polypeptides may on expression in aplant confer a phenotype which is insensitive or unresponsive totreatment of the plant with GA, that is a mutant phenotype which is notovercome or there is no reversion to wild-type phenotype on treatment ofthe plant with GA (though there may be some response in the plant onprovision or depletion of GA).

A further aspect of the present invention provides a nucleic acidisolate having a nucleotide sequence encoding a polypeptide whichincludes an amino acid sequence which is a mutant, allele, derivative orvariant sequence of the GAI amino acid sequence of the speciesArabidopsis thaliana shown in FIG. 4 (SEQ ID NO:2), or is a homologue ofanother species or a mutant, allele, derivative or variant thereof,wherein said mutant, allele, derivative, variant or homologue differsfrom the amino acid sequence shown in FIG. 4 (SEQ ID NO:2) by way ofinsertion, deletion, addition and/or substitution of one or more aminoacids, as obtainable by producing transgenic plants by transformingplants which have a GAI null mutant phenotype, which phenotype isresistance to the dwarfing effect of paclobutrazol, with test nucleicacid, causing or allowing expression from test nucleic acid within thetransgenic plants, screening the transgenic plants for those exhibitingcomplementation of the GAI null mutant phenotype to identify testnucleic acid able to complement the GAI null mutant, deleting fromnucleic acid so identified as being able to complement the GAI nullmutant a nucleotide sequence encoding the 17 amino acid sequenceunderlined in FIG. 4 or a contiguous 17 amino acid sequence in which atleast 10 residues have similarity or identity with the respective aminoacid in the corresponding position in the 17 amino acid sequenceunderlined in FIG. 4, more preferably 11, 12, 13, 14, 15, 16 or 17.

GAI and gai gene homologues may be identified from economicallyimportant monocotyledonous crop plants such as wheat, rice and maize.Although genes encoding the same protein in monocotyledonous anddicotyledonous plants show relatively little homology at the nucleotidelevel, amino acid sequences are conserved.

In public sequence databases we recently identified several ESTsequences that were obtained in random sequencing programmes and sharehomology with GAI. Table 2 gives details, showing that homologoussequences have been found in various species, including Zea Mays(maize), 0. Sativa (rice), and Brassica napus (rape) By sequencing,study of expression patterns and examining the effect of altering theirexpression, GAI gene homologues, carrying out a similar function inother plants, are obtainable. Of course, novel uses and mutants,derivatives and alleles of these sequences are included within the scopeof the various aspects of the present invention in the same terms asdiscussed above for the Arabidopsis thaliana gene.

A cell containing nucleic acid of the present invention represents afurther aspect of the invention, particularly a plant cell, or abacterial cell.

The cell may comprise the nucleic acid encoding the enzyme by virtue ofintroduction into the cell or an ancestor thereof of the nucleic acid,e.g. by transformation using any suitable technique available to thoseskilled in the art.

Also according to the invention there is provided a plant cell havingincorporated into its genome nucleic acid as disclosed. The presentinvention also provides a plant comprising such a plant cell.

Also according to the invention there is provided a plant cell havingincorporated into its genome a sequence of nucleotides as provided bythe present invention, under operative control of a regulatory sequencefor control of expression. A further aspect of the present inventionprovides a method of making such a plant cell involving introduction ofa vector comprising the sequence of nucleotides into a plant cell andcausing or allowing recombination between the vector and the plant cellgenome to introduce the sequence of nucleotides into the genome.

A plant according to the present invention may be one which does notbreed true in one or more properties. Plant varieties may be excluded,particularly registrable plant varieties according to Plant Breeders'Rights. It is noted that a plant need not be considered a “plantvariety” simply because it contains stably within its genome atransgene, introduced into a cell of the plant or an ancestor thereof.

In addition to a plant, the present invention provides any clone of sucha plant, seed, selfed or hybrid progeny and descendants, and any part ofany of these, such as cuttings, seed. The invention provides any plantpropagule, that is any part which may be used in reproduction orpropagation, sexual or asexual, including cuttings, seed and so on. Alsoencompassed by the invention is a plant which is a sexually or asexuallypropagated off-spring, clone or descendant of such a plant, or any partor propagule of said plant, off-spring, clone or descendant.

The invention further provides a method of influencing thecharacteristics of a plant comprising expression of a heterologous GAIor gai gene sequence (or mutant, allele, derivative or homologuethereof, as discussed) within cells of the plant. The term“heterologous” indicates that the gene/sequence of nucleotides inquestion have been introduced into said cells of the plant, or anancestor thereof, using genetic engineering, that is to say by humanintervention, which may comprise transformation. The gene may be on anextra-genomic vector or incorporated, preferably stably, into thegenome. The heterologous gene may replace an endogenous equivalent gene,ie one which normally performs the same or a similar function in controlof growth and/or development, or the inserted sequence may be additionalto an endogenous gene. An advantage of introduction of a heterologousgene is the ability to place expression of the gene under the control ofa promoter of choice, in order to be able to influence gene expression,and therefore growth and/or development of the plant according topreference. Furthermore, mutants and derivatives of the wild-type genemay be used in place of the endogenous gene. The inserted gene may beforeign or exogenous to the host cell, e.g. of another plant species.

The principal characteristic which may be altered using the presentinvention is growth.

According to the model of the GAI gene as a growth repressor,under-expression of the gene may be used to promote growth, at least inplants which have only one endogenous gene conferring GAI function (notfor example Arabidopsis which has endogenous homologues which wouldcompensate). This may involve use of anti-sense or sense regulation.Taller plants may be made by knocking out GAI or the relevant homologousgene in the plant of interest. Plants may be made which are resistant tocompounds which inhibit GA biosynthesis, such as paclobutrazol, forinstance to allow use of a GA biosynthesis inhibitor to keep weeds dwarfbut let crop plants grow tall.

Over-expression of a GAI gene may lead to a dwarf plant which iscorrectable by treatment with GA, as predicted by the GAI repressionmodel.

Since gai mutant genes are dominant on phenotype, they may be used tomake GA-insensitive dwarf plants. This may be applied for example to anytransformable crop-plant, tree or fruit-tree species. It may providehigher yield/reduced lodging like Rht wheat. In rice this may provideGA-insensitive rice resistant to the Bakane disease, which is a problemin Japan and elsewhere. Dwarf ornamentals may be of value for thehorticulture and cut-flower markets. Sequence manipulation may providefor varying degrees of severity of dwarfing, GA-insensitive phenotype,allowing tailoring of the degree of severity to the needs of eachcrop-plant or the wishes of the manipulator. Over-expression ofgai-mutant sequences is potentially the most useful.

A second characteristic that may be altered is plant development, forinstance flowering. In some plants, and in certain environmentalconditions, a GA signal is required for floral induction. For example,GA-deficient mutant Arabidopsis plants grown under short day conditionswill do not flower unless treated with GA: these plants do flowernormally when grown under long day conditions. Arabidopsis gai mutantplants show delayed flowering under short day conditions: severe mutantsmay not flower at all. Thus, for instance by GAI or gal gene expressionor over-expression, plants may be produced which remain vegetative untilgiven GA treatment to induce flowering. This may be useful inhorticultural contexts or for spinach, lettuce and other crops wheresuppression of bolting is desirable.

The nucleic acid according to the invention may be placed under thecontrol of an externally inducible gene promoter to place the GAI or gaicoding sequence under the control of the user.

The term “inducible” as applied to a promoter is well understood bythose skilled in the art. In essence, expression under the control of aninducible promoter is “switched-on” or increased in response to anapplied stimulus. The nature of the stimulus varies between promoters.Some inducible promoters cause little or undetectable levels of expression (or no expression) in the absence of the appropriate stimulus. Otherinducible promoters cause detectable constitutive expression in theabsence of the stimulus. Whatever the level of expression is in theabsence of the stimulus, expression from any inducible promoter isincreased in the presence of the correct stimulus. The preferablesituation is where the level of expression increases upon application ofthe relevant stimulus by an amount effective to alter a phenotypiccharacteristic. Thus an inducible (or “switchable”) promoter may be usedwhich causes a basic level of expression in the absence of the stimuluswhich level is too low to bring about a desired phenotype (and may infact be zero). Upon application of the stimulus, expression is increased(or switched on) to a level which brings about the desired phenotype.

Suitable promoters include the Cauliflower Mosaic Virus 35S (CaMV 35S)gene promoter that is expressed at a high level in virtually all planttissues (Benfey et al, 1990a and 1990b); the maizeglutathione-S-transferase isoform II (GST-II-27) gene promoter which isactivated in response to application of exogenous safener (WO93/01294,ICI Ltd); the cauliflower meri 5 promoter that is expressed in thevegetative apical meristem as well as several well localised positionsin the plant body, eg inner phloem, flower primordia, branching pointsin root and shoot (Medford, 1992; Medford et al, 1991) and theArabidopsis thaliana LEAFY promoter that is expressed very early inflower development (Weigel et al, 1992).

The GST-II-27 gene promoter has been shown to be induced by certainchemical compounds which can be applied to growing plants. The promoteris functional in both monocotyledons and dicotyledons. It can thereforebe used to control gene-expression in a variety of genetically modifiedplants, including field crops such as canola, sunflower, tobacco,sugarbeet, cotton; cereals such as wheat, barley, rice, maize, sorghum;fruit such as tomatoes, mangoes, peaches, apples, pears, strawberries,bananas, and melons; and vegetables such as carrot, lettuce, cabbage andonion. The GST-II-27 promoter is also suitable for use in a variety oftissues, including roots, leaves, stems and reproductive tissues.

Accordingly, the present invention provides in a further aspect a geneconstruct comprising an inducible promoter operatively linked to anucleotide sequence provided by the present invention, such as the GAIgene of Arabidopsis thaliana, a homologue from another plant species orany mutant, derivative or allele thereof. This enables control ofexpression of the gene. The invention also provides plants transformedwith said gene construct and methods comprising introduction of such aconstruct into a plant cell and/or induction of expression of aconstruct within a plant cell, by application of a suitable stimulus, aneffective exogenous inducer. The promoter may be the GST-II-27 genepromoter or any other inducible plant promoter.

When introducing a chosen gene construct into a cell, certainconsiderations must be taken into account, well known to those skilledin the art. The nucleic acid to be inserted should be assembled within aconstruct which contains effective regulatory elements which will drivetranscription. There must be available a method of transporting theconstruct into the cell. Once the construct is within the cell membrane,integration into the endogenous chromosomal material either will or willnot occur. Finally, as far as plants are concerned the target cell typemust be such that cells can be regenerated into whole plants.

Selectable genetic markers may be used consisting of chimaeric genesthat confer selectable phenotypes such as resistance to antibiotics suchas kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate,gentamycin, spectinomycin, imidazolinones and glyphosate.

An aspect of the present invention is the use of nucleic acid accordingto the invention in the production of a transgenic plant.

A further aspect provides a method including introducing the nucleicacid into a plant cell and causing or allowing incorporation of thenucleic acid into the genome of the cell.

Any appropriate method of plant transformation may be used to generateplant cells comprising nucleic acid in accordance with the presentinvention. Following transformation, plants may be regenerated fromtransformed plant cells and tissue.

Successfully transformed cells and/or plants, i.e. with the constructincorporated into their genome, may be selected following introductionof the nucleic acid into plant cells, optionally followed byregeneration into a plant, e.g. using one or more marker genes such asantibiotic resistance (see above).

Plants transformed with the DNA segment containing the sequence may beproduced by standard techniques which are already known for the geneticmanipulation of plants. DNA can be transformed into plant cells usingany suitable technology, such as a disarmed Ti-plasmid vector carried byAgrobacterium exploiting its natural gene transfer ability (EP-A-270355,EP-A-0116718, NAR 12(22) 8711-87215 1984), particle or microprojectilebombardment (U.S. Pat. No. 5,100,792, EP-A-444882, EP-A-434616)microinjection (WO 92/09696, WO 94/00583, EP 331083, EP 175966, Green etal. (1987) Plant Tissue and Cell Culture, Academic Press),electroporation (EP 290395, WO 8706614 Gelvin Debeyser—see attached)other forms of direct DNA uptake (DE 4005152, WO 9012096, U.S. Pat. No.4,684,611), liposome mediated DNA uptake (e.g. Freeman et al. Plant CellPhysiol. 29: 1353 (1984)), or the vortexing method (e.g. Kindle, PNASU.S.A. 87: 1228 (1990d). Physical methods for the transformation ofplant cells are reviewed in Oard, 1991, Biotech. Adv. 9: 1-11.

Agrobacterium transformation is widely used by those skilled in the artto transform dicotyledonous species. Recently, there has beensubstantial progress towards the routine production of stable, fertiletransgenic plants in almost all economically relevant monocot plants(Toriyama, et al. (1988) Bio/Technology 6, 1072-1074; Zhang, et al.(1988) Plant Cell Rep. 7, 379-384; Zhang, et al. (1988) Theor Appl Genet76, 835-840; Shimamoto, et al. (1989) Nature 338, 274-276; Datta, et al.(1990) Bio/Technology 8, 736-740; Christou, et al. (1991) Bio/Technology9, 957-962; Peng, et al. (1991) International Rice Research Institute,Manila, Philippines 563-574; Cao, et al. (1992) Plant Cell Rep. 11,585-591; Li, et al. (1993) Plant Cell Rep. 12, 250-255; Rathore, et al.(1993) Plant Molecular Biology 21, 871-884; Fromm, et al. (1990)Bio/Technology 8, 833-839; Gordon-Kamm, et al. (1990) Plant Cell 2,603-618; D'Halluin, et al. (1992) Plant Cell 4, 1495-1505; Walters, etal. (1992) Plant Molecular Biology 18, 189-200; Koziel, et al. (1993)Biotechnology 11, 194-200; Vasil, I. K. (1994) Plant Molecular Biology25, 925-937; Weeks, et al. (1993) Plant Physiology 102, 1077-1084;Somers, et al. (1992) Bio/Technology 10, 1589-1594; WO92/14828). Inparticular, Agrobacterium mediated transformation is now emerging alsoas an highly efficient transformation method in monocots (Hiei et al.(1994) The Plant Journal 6, 271-282).

The generation of fertile transgenic plants has been achieved in thecereals rice, maize, wheat, oat, and barley (reviewed in Shimamoto, K.(1994) Current Opinion in Biotechnology 5, 158-162; Vasil, et al. (1992)Bio/Technology 10, 667-674; Vain et al., 1995, Biotechnology Advances 13(4): 653-671; Vasil, 1996, Nature Biotechnology 14 page 702).

Microprojectile bombardment, electroporation and direct DNA uptake arepreferred where Agrobacterium is inefficient or ineffective.Alternatively, a combination of different techniques may be employed toenhance the efficiency of the transformation process, eg bombardmentwith Agrobacterium coated microparticles (EP-A-486234) ormicroprojectile bombardment to induce wounding followed byco-cultivation with Agrobacterium (EP-A-486233).

Brassica napus transformation is described in Moloney et al. (1989)Plant Cell Reports 8: 238-242.

Following transformation, a plant may be regenerated, e.g. from singlecells, callus tissue or leaf discs, as is standard in the art. Almostany plant can be entirely regenerated from cells, tissues and organs ofthe plant. Available techniques are reviewd in Vasil et al., CellCulture and Somatic Cel Genetics of Plants, Vol I, II and III,Laboratory Procedures and Their Applications, Academic Press, 1984, andWeissbach and Weissbach, Methods for Plant Molecular Biology, AcademicPress, 1989.

The particular choice of a transformation technology will be determinedby its efficiency to transform certain plant species as well as theexperience and preference of the person practising the invention with aparticular methodology of choice. It will be apparent to the skilledperson that the particular choice of a transformation system tointroduce nucleic acid into plant cells is not essential to or alimitation of the invention, nor is the choice of technique for plantregeneration.

In the present invention, over-expression may be achieved byintroduction of the nucleotide sequence in a sense orientation. Thus,the present invention provides a method of influencing a characteristicof a plant, the method comprising causing or allowing expression ofnucleic acid according to the invention from that nucleic acid withincells of the plant. Under-expression of the gene product polypeptide maybe achieved using anti-sense technology or “sense regulation”. The useof anti-sense genes or partial gene sequences to down-regulate geneexpression is now well-established. DNA is placed under the control of apromoter such that transcription of the “anti-sense” strand of the DNAyields RNA which is complementary to normal mRNA transcribed from the“sense” strand of the target gene. For double-stranded DNA this isachieved by placing a coding sequence or a fragment thereof in a“reverse orientation” under the control of a promoter. The complementaryanti-sense RNA sequence is thought then to bind with mRNA to form aduplex, inhibiting translation of the endogenous mRNA from the targetgene into protein. Whether or not this is the actual mode of action isstill uncertain. However, it is established fact that the techniqueworks. See, for example, Rothstein et al, 1987; Smith et al, (1988)Nature 334, 724-726; Zhang et al, (1992) The Plant Cell 4, 1575-1588,English et al., (1996) The Plant Cell 8, 179-188. Antisense technologyis also reviewed in reviewed in Bourque, (1995), Plant Science 105,125-149, and Flavell, (1994) PNAS USA 91, 3490-3496.

The complete sequence corresponding to the coding sequence in reverseorientation need not be used. For example fragments of sufficient lengthmay be used. It is a routine matter for the person skilled in the art toscreen fragments of various sizes and from various parts of the codingsequence to optimise the level of anti-sense inhibition. It may beadvantageous to include the initiating methionine ATG codon, and perhapsone or more nucleotides upstream of the initiating codon. A furtherpossibility is to target a regulatory sequence of a gene, e.g. asequence that is characteristic of one or more genes in one or morepathogens against which resistance is desired. A suitable fragment mayhave at least about 14-23 nucleotides, e.g. about 15, 16 or 17, or more,at least about 25, at least about 30, at least about 40, at least about50, or more. Such fragments in the sense orientation may be used inco-suppression (see below).

Total complementarity of sequence is not essential, though may bepreferred. One or more nucleotides may differ in the anti-senseconstruct from the target gene. It may be preferred for there to besufficient homology for the respective anti-sense and sense RNAmolecules to hybridise, particularly under the conditions existing in aplant cell.

Thus, the present invention also provides a method of influencing acharacteristic of a plant, the method comprising causing or allowinganti-sense transcription from nucleic acid according to the inventionwithin cells of the plant.

When additional copies of the target gene are inserted in sense, that isthe same, orientation as the target gene, a range of phenotypes isproduced which includes individuals where over-expression occurs andsome where under-expression of protein from the target gene occurs. Whenthe inserted gene is only part of the endogenous gene the number ofunder-expressing individuals in the transgenic population increases. Themechanism by which sense regulation occurs, particularlydown-regulation, is not well-understood. However, this technique is alsowell-reported in scientific and patent literature and is used routinelyfor gene control. See, for example, See, for example, van der Krol etal., (1990) The Plant Cell 2, 291-299; Napoli et al., (1990) The PlantCell 2, 279-289; Zhang et al., (1992) The Plant Cell 4, 1575-1588, andU.S. Pat. No. 5,231,020.

Thus, the present invention also provides a method of influencing acharacteristic of a plant, the method comprising causing or allowingexpression from nucleic acid according to the invention within cells ofthe plant. This may be used to influence growth.

Aspects and embodiments of the present invention will now beillustrated, by way of example, with reference to the accompanyingfigures. Further aspects and embodiments will be apparent to thoseskilled in the art. All documents mentioned in this text areincorporated herein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The following Figures are included herein:

FIG. 1: The basic carbon-ring structure of gibberellins.

FIGS. 2(A-C): The gai-t6 line contains a transposed Ds which interruptsa transcribed gene.

FIG. 2a: Plants shown are (left to right) homozygous for GAI, gai andgai-t6. GAI and gai-t6 plants are indistinguishable.

FIG. 2b: DNA gel-blot hybridization using a Ds probe. DNA in the GAIlane lacks Ds. The gai lane contains DNA from plants homozygous for galand for T-DNA A264⁵, which contains Ds (18.0 kb EcoRI fragment). Thegai-t6 lane contains DNA from plants homozygous for A264 and for atransposed Ds (15.5 kb fragment).

FIG. 2c: DNA gel-blot hybridization using a radiolabelled GAI cDNAprobe. The cDNA hybridizes with a 5.1 kb Bc1I fragment in DNA from GAIand gai, replaced in gai-t6 by fragments of 6.4 and 2.8 kb. Since Bc1Icuts once within Ds, the Ds insertion is flanked on either side by thegene (GAI) encoding the cDNA. The fainter hybridization at 17 kb is oneof several seen on longer exposure and identifies a sequence related toGAI.

FIG. 3: A nucleotide sequence of (SEQ ID NO:1) a GAI gene encoding apolypeptide with GAI function.

FIG. 4: Primary structure of GAI and gai proteins. The amino acidsequence (SEQ ID NO:2) predicted from the genomic DNA sequence of GAI isshown. The 17 amino acid segment deleted in gai is shown in bold faceand double-underlined.

FIG. 5: De-repression model for plant growth regulation by GA.

FIG. 6(A-H): Nucleotide and encoded amino acid sequences ofgai-derivative alleles.

FIG. 6a: Nucleotide sequence of gai-d1 (SEQ ID NO:3).

FIG. 6b: Amino acid sequence of gai-d1 (SEQ ID NO:4).

FIG. 6c: Nucleotide sequence of gai-d2 (SEQ ID NO:5).

FIG. 6d: Amino acid sequence of gai-d2 (SEQ ID NO:6).

FIG. 6e: Nucleotide sequence of gai-d5 (SEQ ID NO:7).

FIG. 6f: Amino acid sequence of gai-d5 (SEQ ID NO:8).

FIG. 6g: Nucleotide sequence of gai-d7 (SEQ ID NO:9).

FIG. 6h: Amino acid sequence of gai-d7 (SEQ ID NO:10).

DETAILED DESCRIPTION OF THE INVENTION EXAMPLE 1

Cloning of and Characterisation of GAI and gai Genes

gai maps to chromosome 12 of Arabidopsis, approximately 11 cM from aT-DNA insertion carrying a Ds transposon^(5,15). Genetic analysessuggested that loss-of-function alleles confer a tall phenotypeindistinguishable from that conferred by the wild-type allele(GAI)^(5,6). We attempted to clone GAI via insertional mutagenesis,exploiting the tendency of Ds to transpose preferentially to linkedsites^(16,17).

Plant lines homozygous for A264 and gai, containing a transgene(ΔNaeI-sAc(GUS)-1) expressing Ac transposase were constructed. Plantshomozygous for a putative Ds insertion allele, which we designatedgai-t6, were isolated from this material as follows⁵. The material wasbulked up, by self-pollination, over several generations. During thisbulking, searches were made for plants which had stem branches moreelongated than expected for a gai homozygote. Seeds obtained fromself-pollination of such branches were planted out for closerexamination. The progeny of one such branch segregated plants, at afrequency of approximately one quarter, displaying a tall phenotypeindistinguishable from that conferred by GAI (FIG. 2a). These plantswere homozygous for a new gai allele, which we designated gai-t6.

DNA gel-blot experiments revealed that gai-t6 contains a transposed Ds(FIG. 2b), inserted within a region (approximately 200 kb) of chromosome1 known to contain GAS (data not shown). Genomic DNA preparation andgel-blot hybridizations were performed as described⁵. EcoRI digests werehybridized with the Ds probe (radiolabelled 3.4 kb XhoI-BamHIsubfragment of Ac). gai-t6 has lost (ΔNaeI-sAc(GUS)-1) via geneticsegregation.

Further experiments showed that the transposed Ds interrupts thetranscribed region of a gene (GAI), and that the Arabidopsis genomecontains at least one additional gene sharing significant sequencehomology with GAI (FIG. 2c). A radiolabelled IPCR fragment containinggenomic DNA adjacent to the 3′ end of the transposed Ds in gai-t6 wasisolated as previously described²⁴. It was necessary to use considerablecaution in the use of this probe since it was potentially contaminatedwith sequence derived from the T-DNA 3′ of the Ds in A264 (which isstill present in the gai-t6 line): However, the fact that the probehybridized with DNA from plants lacking any T-DNA insertion indicatedthat it was useful for the purposes of cloning the region of genomic DNAinto which the transposed Ds in gai-t6 had inserted. This probe wasshown to hybridize to genomic DNA cosmid clones previously identified asbeing likely to contain GAI by map-based cloning. One of these cosmidswas used to identify, by hybridization, clones from a cDNA library madefrom mRNA isolated from aerial plant parts (Arabidopsis). These cDNAswere classified according to their hybridization to genomic DNA fromGAI, gai and gai-t6. Some of these clones hybridized weakly fragmentscontaining GAI (as defined by the alteration in fragment size caused bythe Ds insertion in gai-t6), but more strongly to other, relatedsequencs. These cDNAs are presumably derived from mRNAs transcribed fromgenes related in sequence to GAI, but not from GAI itself, and were putto one side for future investigations. One cDNA, pPC1, hybridizedstrongly to GAI, and less strongly to the fragments containing sequencerelated to GAI. The DNA sequence of part this cDNA was identical withapproximately 150 bp of genomic DNA flanking the Ds insertion in gai-t6.

Reversion analysis showed that excision of Ds from gai-t6 was associatedwith restoration of a dominant dwarf phenotype.

The DNA sequences of two overlapping GAI cDNAs revealed an open readingframe (ORF) encoding a protein (GAI) of 532 amino acid residues. DNAfragments containing this ORF were amplified from GAI and gai genomicDNA. Oligonucleotide primers derived from the DNA sequences ofoverlapping cDNAs pPC1 and pPC2 were used to amplify, via PCR, 1.7 kbfragments from GAI and gai genomic DNA. The sequences of the primersused were:

Primer N6 (SEQ ID NO:11): 5′TAG AAG TGG TAG TGG3′;

Primer AT1 (SEQ ID NO:12): 5′ACC ATG AGA CCA GCC G3′.

The sequence of primer AT1 differs by one base from the sequence of thegenomic and c-DNA clones. The primer was synthesized very early in thesequencing project, before the final corrected version of the sequencewas available.

The DNA sequences of fragments from duplicate amplifications weredetermined, thus avoiding errors introduced by PCR.

The GAI genomic sequence was almost identical with that of theoverlapping cDNAs. There were three nucleotide substitutions that couldbe due to differences between ecotypes and which do not alter thepredicted amino acid sequence of GAI. The sequences of these genomicfragments revealed that the ORF is not interrupted by introns (FIG. 3).

The Ds insertion in gai-t6 is located between the Glu¹⁸² and Asn¹⁸³codons (FIG. 4). The predicted secondary structure of GAI shows fewsalient features. GAI is a largely hydrophilic protein with apolyhistidine tract of unknown significance close to the amino-terminus,and a weakly hydrophobic domain surrounding a possible glycosylationsite at Asn¹⁸³. Computer analysis indicates a relatively low likelihoodthat this hydrophobic region is a transmembrane domain.

Searches of the DNA and protein sequence databases revealed no domainsof obvious functional significance within GAI. gai contains a deletionof 51 bp from within the GAI ORF. This in-frame deletion results in theabsence, in gai, of a 17 amino acid residue segment situated close tothe amino terminus of the predicted GAI protein (SEQ ID NO:2) (FIG. 4).

Laurenzio et al.⁴⁵ reported after the priority date of the presentinvention a sequence for the SCR (SCARECROW) gene of Arabidopsis,mutation of which results in roots that are missing one cell layer. Thedisclosed SCR sequence has some homology with the Arabidopsis GAIsequence of the present invention, but lacks the 17 amino acid motifdiscussed.

A previous publication described the isolation, following γ-irradiationmutagenesis, of gal derivative alleles⁵. These alleles, when homozygous,confer a tall phenotype indistinguishable from that conferred by GAI⁵.Sequencing of amplified fragments from several of the derivative alleles(gai-di, gai-d2, gai-d5 and gai-d7) showed that each contains the 51 bpdeletion characteristic of gai. Nucleotide and encoded amino acidsequences of these alleles are shown in FIG. 6 (SEQ ID NOS:3 to SEQ IDNO:10). They also contain additional mutations that could confer anon-functional gene product (Table 1). The fact that loss of gai mutantphenotype is correlated with each of these mutations, together with thereversion data (see above), confirms that GAI has been cloned.Furthermore, these results are consistent with predictions that thegai-d alleles would be null alleles^(5,6).

Cloning of gai via insertional mutagenesis was possible because it is again-of-function mutation. Such mutations can have dominant effects fora variety of reasons, including ectopic or increased expression of anormal gene product, or altered function of a mutant gene product. Herewe show that the gai mutation is associated with an altered product.Deletion of a 17 amino acid residue domain from GAI results in a mutantprotein (gai) which, in a genetically dominant fashion, causes dwarfism.This strongly suggests that GAI is a growth repressor, and that GAde-represses growth by antagonizing GAI action. The domain missing inthe mutant gai protein may be responsible for interacting with the GAsignal or with GA itself. gai would then constitutively repress growthbecause it cannot be antagonized by GA. A de-repression model forGA-mediated plant growth regulation is further elaborated in FIG. 5, butit should be noted that this proposal is not to be taken to limit thescope of the present invention. Knowledge of the actual mode of actionof GAI and gai, i.e. how they work, is not a pre-requisite for operationof the present invention, which is founded on cloning of wild-type andmutant versions of the GAI gene.

Mutations at the SPINDLY (SPY) locus of Arabidopsis confer increasedresistance to GA biosynthesis inhibitors and a reduced dependence on GAfor growth regulation¹⁸, phenotypes characteristic of the slendermutants previously described in other plant species¹⁹⁻²³. Recentexperiments have shown that the dwarf phenotype conferred by gai can bepartially suppressed by mutations at SPY and at other loci^(6,9). Wepropose, again without limiting the scope of the present invention, thatSPY, together with proteins encoded by these other loci, is involvedwith the downstream transduction of the growth repressing signal thatoriginates with GAI (FIG. 5).

According to the model shown in FIG. 5, GA de-represses plant growthbecause it (or a GA signalling component) antagonizes the activity ofGAI, a protein which represses growth. The growth repressing signal istransmitted via Spy^(6,18), GAR2⁶, GAS2 (J.P. and N.P.H., unpublished)and other proteins. Normal plants (GAI) grow tall because the level ofendogenous GA is sufficiently high to substantially antagonize theactivity of the GAI repressor. GA-deficient plants contain insufficientGA to antagonize GAI repression to the same degree, and are thusdwarfed²⁵⁻²⁷. gai mutant plants are dwarfed² because the mutant gaiprotein is not antagonized by GA, and represses growth in a dominantfashion. spy, gar2 and gas2 mutations partially suppress gai phenotype,and confer resistance to GA biosynthesis inhibitors^(6,18). Pairwisecombinations of these three mutations confer more extreme gaisuppression and resistance to GA biosynthesis inhibition than isconferred by any of spy, gar2 or gas2 alone. Thus, these genes areproposed to encode downstream components that are responsible for thetransmission of the growth repressing signal from GAI. It is possiblethat the gai mutation is a functional homologue of the GA-insensitivitymutations in maize¹⁰⁻¹² and wheat¹³. Thus this model can be used toprovide a general explanation for the regulation of plant growth by GA.

Independent studies of GA-insensitive dwarf mutants in maize^(11,12),and GA-independent slender mutants in pea and barley¹⁹⁻²³, havepreviously implicated the involvement of a repressor function in GAsignal-transduction. The indications from the worked desribed herein arethat in all probability Arabidopsis GAI is such a repressor. Animportant implication of this is that GA then regulates plant growth notvia activation but by de-repression.

EXAMPLE 2

Cloning of GAI Homologues from Wheat, Rice and Brassica sps

DNA containing potential GAI homologues are isolated from wheat, riceand Brassica by reduced stringency probing of cDNA or genomic DNAlibraries containing DNA from these species. Hybridizing clones are thenpurified using standard techniques.

Alternatively, potential GAI homologues are identified by screening ofEST databases for cDNA and other sequences showing statisticallysignificant homology with the GAI sequence. Clones are then obtained byrequesting them from the relevant distribution centres. Table 2 givesdetails of results of searching in public sequence databases containingEST sequences that were obtained in random sequencing programmes,showing that homologous sequences have been found in various species,including Zea Mays (maize), O. Sativa (rice), and Brassica napus (rape).

In the case of wheat and maize, it is important to know if thesehomologous sequences correspond to the previously characterized Rht andD8 genetic loci. This is determined as follows.

cDNA or genomic DNA from rice, wheat or maize is mapped onto the wheatgenomic map, thus determining if the map position of the DNA correspondsto the map position of the Rht loci in wheat. Furthermore, in the caseof maize, potential transposon-insertion alleles of D8 exist, and theseare used to prove the cloning of D8 in the same manner as we have proventhe cloning of gai from Arabidopsis. By sequencing these various cDNAand genomic DNA clones, studying their expression patterns and examiningthe effect of altering their expression, genes carrying out a similarfunction to GAI in regulating plant growth are obtained.

Mutants, derivatives, variants and alleles of these sequences are madeand identified as appropriate.

EXAMPLE 3

Expression of GAI and gai Proteins in E. coli

DNA fragments containing the complete GAI or gai open reading frameswere amplified using PCR from genomic DNA clones (no introns in gene)containing the GAI and gai genes. Amplifications were done using primerswhich converted the ATG translation start codon into a BamHI restrictionendonuclease site. The fragments have a PstI restriction endonucleasesite at the other end (beyond the stop codon). The products were clonedand their DNA sequences determined to ensure that no errors had beenintroduced during the course of the PCR. The correct fragments werecloned into BamHI/PstI digested PQE30 expression vector(Qiaexpressionist kit from the Qiagen Company), resulting in constructswith the potential to express the GAI and gai proteins in E. coli.Expression in this vector is regulated by an IPTG-inducible promoter,and the resultant proteins carry an N-terminal polyhistidine tag whichcan be used to purify them from cellular extracts.

Induction with IPTG resulted in high-level expression of the GAI and gaiproteins in E. coli.

EXAMPLE 4

Expression Constructs and Transformation of Plants

(a) Normal Expression Levels, Using Endogenous Promoters

The GAI and gai genes were isolated as 5 kb EcoRI/EcoRV fragments(containing about 1.5 kb of non-coding sequence flanking the codingsequence) by subcloning from appropriate genomic clones. These fragmentswere cloned into the Bluescript vector, re-isolated as EcoRI/XbaIfragments, and ligated into binary vectors for mobilisation intoAgrobacterium tumefaciens C58C1, with the T-DNA being introduced intoArabidopsis and tobacco plants as described by Valvekens et al.³² or bythe more recent vacuum infiltration method³³, and into Brassica napususing the high efficiency Agrobacterium transformation technique asdescribed in Moloney et al.³⁴.

(b) Overexpression Using an Exogenous Promoter

Constructs have been made using DNA from vectors pJIT60, containing adouble 35S promoter³⁵ and pJIT62, a modified form of pJIT60 thatcontains a single 35S promoter. The promoters from these vectors werefused with around 100 bp 5′ non-coding sequence, followed by an ATG andthe entire GAI or gai open reading frames, followed by a translationalstop codon, followed by around 20 bp 3′ non-coding sequence, followed bya polyadenylation signal: all this carried on a SstI/XhoI fragment.

This fragment has been ligated into binary vectors for introduction intotransgenic plants, either by the use of Agrobacterium tumefaciens or asnaked DNA, as described earlier.

EXAMPLE 4

Modification of GAI and gai Sequences

A short segment of the GAI open reading frame surrounding the gaideletion is amplified from GAI and gai by using in PCR appropriateoligonucleotide primers, designed on the basis of sequence informationprovided herein. The amplified segment is then subjected to one or moreof various forms of mutagenesis (see e.g. Sambrook et al.), resulting ina series of overlapping deletion mutants, or, if desired, substitutionsof individual nucleotides in this region.

The mutated amplified segment is then substituted for the equivalentsegment in GAI, via restriction endonuclease digestion and a subsequentligation reaction. This new variant is then expressed in transgenicplants either at normal levels or via overexpression as described above.

Constructs are studied to assess their effects on plant growthregulation in model (e.g. Arabidopsis and tobacco) and crop (e.g. wheat,rice and maize) species. Different constructs confer differing degreesof dwarfism and may individually be especially suited to themodification and improvement of particular crop species, or for cropsgrowing in particular environments.

EXAMPLE 5

GAI Null Alleles Confer Increased Resistance to Paclobutrazol:

Paclobutrazol is a triazole derivative that specifically inhibits GAbiosynthesis at the kaurene oxidase reaction^(36,37), thus reducingendogenous GA levels and conferring a dwarf phenotype on plants exposedto it. The slender mutants of pea and barley are resistant to thedwarfing effects of paclobutrazol³⁸⁻⁴², as is the Arabidopsisconstitutive GA-response mutant spy^(43,44). Thus, in these mutants stemelongation is at least partially uncoupled from the GA-mediated controlcharacteristic of normal plants. Interestingly, the gai-t6 mutant alsodisplays paclobutrazol resistance. When grown on medium containingpaclobutrazol, gai-t6 mutants display longer floral bolt stems than GAIcontrol plants. This result suggests that loss of GAI function causes areduction in the GA-dependency of stem elongation. Put another way, aGAI null mutant appears to require less endogenous GA to achieve acertain degree of growth than does a normal plant. GA-dependency is notcompletely abolished by gai-t6 possibly because the products of genesrelated in sequence to GAI (see above) can substantially, but notcompletely, compensate for loss of GAI function. These observations aresignificant, because they demonstrate that the wild-type gene product,GAI, is a GA signal-transduction component.

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TABLE 1 Mutations in GAI alleles Position in Nature of CodingConsequence of Allele Mutation* Sequence Mutation gai-d1 CAG to TAGGlu²³⁹ Stop codon, truncated polypeptide gai-d2 GAT to GA, Asp²⁷⁴Frameshift, addition of one base two novel amino acids, deletiontruncated polypeptide gai-d5 7 base follows Leu²⁸¹ Frameshift, additionof deletion, 18 novel amino acids, also C to G truncated polypeptidegai-d7 GTT to GT, Val¹⁵⁶ Frameshift, addition of one base 27 novel aminoacids, deletion truncated polypeptide *Underlining denotes nucleotidesubstitution in each allele. The alleles were isolated followingγ-irradiation mutagenesis of gai homozygotes⁵. 1.7 kb fragments wereamplified from genomic DNA from each allele, and sequenced as describedabove. Each allele contains the 51 bp deletion characteristic of gai,confirming that they are all genuinely derived from gai and are notcontaminants.

TABLE 2 Databases searched on 11/1/96 ESTs with homology to the GAIc-DNA Blast Poisson Clone ID Species probability 1.- HOMOLOGY TO THEFIRST 200 AMINOACIDS. EM_EST1:ATTS3217 A. Thaliana 4.8 · e⁻³²EM_EST1:AT7823 A. Thaliana 4.8 · e⁻²⁴ EM_EST1:AT7938 A. Thaliana 7.2 ·e⁻²² EM_EST3:OSS0803A O. Sativa (rice) 7.8 · e⁻¹¹ EM_EST1:AT5178 A.Thaliana 0.014 EM_EST1:AT9456 A. Thaliana 0.026 2.- HOMOLOGY TOAMINOACIDS 200-400. EM_EST1:ATTS4818 A. Thaliana 1.5 · e⁻²¹EM_EST3:ZM3101 Zea Mays (maize) 9.1 · e⁻¹⁴ EM_EST1:ATTS1110 A. Thaliana7.9 · e⁻¹⁰ EM_EST1:ATTS3935 A. Thaliana 1.7 · e⁻⁹ EM_STS:ZM7862 Zea Mays(maize) 4.5 · e⁻⁷ EM_EST1:AT7938 A. Thaliana 0.00011 EM_EST3:OSS3989A O.Sativa (rice) 0.00050 3.- HOMOLOGY TO THE LAST 132 AMINOACIDS.EM_EST1:AT2057 A. Thaliana 3.1 · e⁻⁵² EM_EST1:ATTS3359 A. Thaliana 3.2 ·e⁻⁴² EM_EST3:OSO713A O. Sativa (rice) 2.8 · e⁻¹⁰ EM_EST1:BN6691 B. Napus(rape) 3.0 · e⁻⁵ EM_EST1:ATTS3934 A. Thaliana 0.00034 EM_EST1:ATTS4819A. Thaliana 0.00059 EM_EST1:AT4839 A. Thaliana 0.00060 EM_EST1:ATTS1327A. Thaliana 0.00073 EM_EST1:AT1868 A. Thaliana 0.0054 EM_EST1:AT79316 A.Thaliana O.092 EM_EST1:AT7747 A. Thaliana 0.35

12 1 1964 DNA Arabidopsis thaliana 1 taataatcat tttttttctt ataaccttcctctctatttt tacaatttat tttgttatta 60 gaagtggtag tggagtgaaa aaacaaatcctaagcagtcc taaccgatcc ccgaagctaa 120 agattcttca ccttcccaaa taaagcaaaacctagatccg acattgaagg aaaaaccttt 180 tagatccatc tctgaaaaaa aaccaaccatgaagagagat catcatcatc atcatcaaga 240 taagaagact atgatgatga atgaagaagacgacggtaac ggcatggatg agcttctagc 300 tgttcttggt tacaaggtta ggtcatcggaaatggctgat gttgctcaga aactcgagca 360 gcttgaagtt atgatgtcta atgttcaagaagacgatctt tctcaactcg ctactgagac 420 tgttcactat aatccggcgg agctttacacgtggcttgat tctatgctca ccgaccttaa 480 tcctccgtcg tctaacgccg agtacgatcttaaagctatt cccggtgacg cgattctcaa 540 tcagttcgct atcgattcgg cttcttcgtctaaccaaggc ggcggaggag atacgtatac 600 tacaaacaag cggttgaaat gctcaaacggcgtcgtggaa accaccacag cgacggctga 660 gtcaactcgg catgttgtcc tggttgactcgcaggagaac ggtgtgcgtc tcgttcacgc 720 gcttttggct tgcgctgaag ctgttcagaaggagaatctg actgtggcgg aagctctggt 780 gaagcaaatc ggattcttag ctgtttctcaaatcggagct atgagaaaag tcgctactta 840 cttcgccgaa gctctcgcgc ggcggatttaccgtctctct ccgtcgcaga gtccaatcga 900 ccactctctc tccgatactc ttcagatgcacttctacgag acttgtcctt atctcaagtt 960 cgctcacttc acggcgaatc aagcgattctcgaagctttt caagggaaga aaagagttca 1020 tgtcattgat ttctctatga gtcaaggtcttcaatggccg gcgcttatgc aggctcttgc 1080 gcttcgacct ggtggtcctc ctgttttccggttaaccgga attggtccac cggcaccgga 1140 taatttcgat tatcttcatg aagttgggtgtaagctggct catttagctg aggcgattca 1200 cgttgagttt gagtacagag gatttgtggctaacacttta gctgatcttg atgcttcgat 1260 gcttgagctt agaccaagtg agattgaatctgttgcggtt aactctgttt tcgagcttca 1320 caagctcttg ggacgacctg gtgcgatcgataaggttctt ggtgtggtga atcagattaa 1380 accggagatt ttcactgtgg ttgagcaggaatcgaaccat aatagtccga ttttcttaga 1440 tcggtttact gagtcgttgc attattactcgacgttgttt gactcgttgg aaggtgtacc 1500 gagtggtcaa gacaaggtca tgtcggaggtttacttgggt aaacagatct gcaacgttgt 1560 ggcttgtgat ggacctgacc gagttgagcgtcatgaaacg ttgagtcagt ggaggaaccg 1620 gttcgggtct gctgggtttg cggctgcacatattggttcg aatgcgttta agcaagcgag 1680 tatgcttttg gctctgttca acggcggtgagggttatcgg gtggaggaga gtgacggctg 1740 tctcatgttg ggttggcaca cacgaccgctcatagccacc tcggcttgga aactctccac 1800 caattagatg gtggctcaat gaattgatctgttgaaccgg ttatgatgat agatttccga 1860 ccgaagccaa actaaatcct actgtttttccctttgtcac ttgttaagat cttatctttc 1920 attatattag gtaattgaaa aatttctaaattactcacac tggc 1964 2 532 PRT Arabidopsis thaliana 2 Met Lys Arg AspHis His His His His Gln Asp Lys Lys Thr Met Met 1 5 10 15 Met Asn GluGlu Asp Asp Gly Asn Gly Met Asp Glu Leu Leu Ala Val 20 25 30 Leu Gly TyrLys Val Arg Ser Ser Glu Met Ala Asp Val Ala Gln Lys 35 40 45 Leu Glu GlnLeu Glu Val Met Met Ser Asn Val Gln Glu Asp Asp Leu 50 55 60 Ser Gln LeuAla Thr Glu Thr Val His Tyr Asn Pro Ala Glu Leu Tyr 65 70 75 80 Thr TrpLeu Asp Ser Met Leu Thr Asp Leu Asn Pro Pro Ser Ser Asn 85 90 95 Ala GluTyr Asp Leu Lys Ala Ile Pro Gly Asp Ala Ile Leu Asn Gln 100 105 110 PheAla Ile Asp Ser Ala Ser Ser Ser Asn Gln Gly Gly Gly Gly Asp 115 120 125Thr Tyr Thr Thr Asn Lys Arg Leu Lys Cys Ser Asn Gly Val Val Glu 130 135140 Thr Thr Thr Ala Thr Ala Glu Ser Thr Arg His Val Val Leu Val Asp 145150 155 160 Ser Gln Glu Asn Gly Val Arg Leu Val His Ala Leu Leu Ala CysAla 165 170 175 Glu Ala Val Gln Lys Glu Asn Leu Thr Val Ala Glu Ala LeuVal Lys 180 185 190 Gln Ile Gly Phe Leu Ala Val Ser Gln Ile Gly Ala MetArg Lys Val 195 200 205 Ala Thr Tyr Phe Ala Glu Ala Leu Ala Arg Arg IleTyr Arg Leu Ser 210 215 220 Pro Ser Gln Ser Pro Ile Asp His Ser Leu SerAsp Thr Leu Gln Met 225 230 235 240 His Phe Tyr Glu Thr Cys Pro Tyr LeuLys Phe Ala His Phe Thr Ala 245 250 255 Asn Gln Ala Ile Leu Glu Ala PheGln Gly Lys Lys Arg Val His Val 260 265 270 Ile Asp Phe Ser Met Ser GlnGly Leu Gln Trp Pro Ala Leu Met Gln 275 280 285 Ala Leu Ala Leu Arg ProGly Gly Pro Pro Val Phe Arg Leu Thr Gly 290 295 300 Ile Gly Pro Pro AlaPro Asp Asn Phe Asp Tyr Leu His Glu Val Gly 305 310 315 320 Cys Lys LeuAla His Leu Ala Glu Ala Ile His Val Glu Phe Glu Tyr 325 330 335 Arg GlyPhe Val Ala Asn Thr Leu Ala Asp Leu Asp Ala Ser Met Leu 340 345 350 GluLeu Arg Pro Ser Glu Ile Glu Ser Val Ala Val Asn Ser Val Phe 355 360 365Glu Leu His Lys Leu Leu Gly Arg Pro Gly Ala Ile Asp Lys Val Leu 370 375380 Gly Val Val Asn Gln Ile Lys Pro Glu Ile Phe Thr Val Val Glu Gln 385390 395 400 Glu Ser Asn His Asn Ser Pro Ile Phe Leu Asp Arg Phe Thr GluSer 405 410 415 Leu His Tyr Tyr Ser Thr Leu Phe Asp Ser Leu Glu Gly ValPro Ser 420 425 430 Gly Gln Asp Lys Val Met Ser Glu Val Tyr Leu Gly LysGln Ile Cys 435 440 445 Asn Val Val Ala Cys Asp Gly Pro Asp Arg Val GluArg His Glu Thr 450 455 460 Leu Ser Gln Trp Arg Asn Arg Phe Gly Ser AlaGly Phe Ala Ala Ala 465 470 475 480 His Ile Gly Ser Asn Ala Phe Lys GlnAla Ser Met Leu Leu Ala Leu 485 490 495 Phe Asn Gly Gly Glu Gly Tyr ArgVal Glu Glu Ser Asp Gly Cys Leu 500 505 510 Met Leu Gly Trp His Thr ArgPro Leu Ile Ala Thr Ser Ala Trp Lys 515 520 525 Leu Ser Thr Asn 530 31643 DNA Arabidopsis thaliana 3 tagaagtggt agtggagtga aaaaacaaatcctaagcagt cctaaccgat ccccgaagct 60 aaagattctt caccttccca aataaagcaaaacctagatc cgacattgaa ggaaaaacct 120 tttagatcca tctctgaaaa aaaaccaaccatgaagagag atcatcatca tcatcatcaa 180 gataagaaga ctatgatgat gaatgaagaagacgacggta acggcatgga tgttgctcag 240 aaactcgagc agcttgaagt tatgatgtctaatgttcaag aagacgatct ttctcaactc 300 gctactgaga ctgttcacta taatccggcggagctttaca cgtggcttga ttctatgctc 360 accgacctta atcctccgtc gtctaacgccgagtacgatc ttaaagctat tcccggtgac 420 gcgattctca atcagttcgc tatcgattcggcttcttcgt ctaaccaagg cggcggagga 480 gatacgtata ctacaaacaa gcggttgaaatgctcaaacg gcgtcgtgga aaccaccaca 540 gcgacggctg agtcaactcg gcatgttgtcctggttgact cgcaggagaa cggtgtgcgt 600 ctcgttcacg cgcttttggc ttgcgctgaagctgttcaga aggagaatct gactgtggcg 660 gaagctctgg tgaagcaaat cggattcttagctgtttctc aaatcggagc tatgagaaaa 720 gtcgctactt acttcgccga agctctcgcgcggcggattt accgtctctc tccgtcgcag 780 agtccaatcg accactctct ctccgatactctttagatgc acttctacga gacttgtcct 840 tatctcaagt tcgctcactt cacggcgaatcaagcgattc tcgaagcttt tcaagggaag 900 aaaagagttc atgtcattga tttctctatgagtcaaggtc ttcaatggcc ggcgcttatg 960 caggctcttg cgcttcgacc tggtggtcctcctgttttcc ggttaaccgg aattggtcca 1020 ccggcaccgg ataatttcga ttatcttcatgaagttgggt gtaagctggc tcatttagct 1080 gaggcgattc acgttgagtt tgagtacagaggatttgtgg ctaacacttt agctgatctt 1140 gatgcttcga tgcttgagct tagaccaagtgagattgaat ctgttgcggt taactctgtt 1200 ttcgagcttc acaagctctt gggacgacctggtgcgatcg ataaggttct tggtgtggtg 1260 aatcagatta aaccggagat tttcactgtggttgagcagg aatcgaacca taatagtccg 1320 attttcttag atcggtttac tgagtcgttgcattattact cgacgttgtt tgactcgttg 1380 gaaggtgtac cgagtggtca agacaaggtcatgtcggagg tttacttggg taaacagatc 1440 tgcaacgttg tggcttgtga tggacctgaccgagttgagc gtcatgaaac gttgagtcag 1500 tggaggaacc ggttcgggtc tgctgggtttgcggctgcac atattggttc gaatgcgttt 1560 aagcaagcga gtatgctttt ggctctgttcaacggcggtg agggttatcg ggtggaggag 1620 agtgacggct gtctcatgtt ggg 1643 4221 PRT Arabidopsis thaliana 4 Met Lys Arg Asp His His His His His GlnAsp Lys Lys Thr Met Met 1 5 10 15 Met Asn Glu Glu Asp Asp Gly Asn GlyMet Asp Val Ala Gln Lys Leu 20 25 30 Glu Gln Leu Glu Val Met Met Ser AsnVal Gln Glu Asp Asp Leu Ser 35 40 45 Gln Leu Ala Thr Glu Thr Val His TyrAsn Pro Ala Glu Leu Tyr Thr 50 55 60 Trp Leu Asp Ser Met Leu Thr Asp LeuAsn Pro Pro Ser Ser Asn Ala 65 70 75 80 Glu Tyr Asp Leu Lys Ala Ile ProGly Asp Ala Ile Leu Asn Gln Phe 85 90 95 Ala Ile Asp Ser Ala Ser Ser SerAsn Gln Gly Gly Gly Gly Asp Thr 100 105 110 Tyr Thr Thr Asn Lys Arg LeuLys Cys Ser Asn Gly Val Val Glu Thr 115 120 125 Thr Thr Ala Thr Ala GluSer Thr Arg His Val Val Leu Val Asp Ser 130 135 140 Gln Glu Asn Gly ValArg Leu Val His Ala Leu Leu Ala Cys Ala Glu 145 150 155 160 Ala Val GlnLys Glu Asn Leu Thr Val Ala Glu Ala Leu Val Lys Gln 165 170 175 Ile GlyPhe Leu Ala Val Ser Gln Ile Gly Ala Met Arg Lys Val Ala 180 185 190 ThrTyr Phe Ala Glu Ala Leu Ala Arg Arg Ile Tyr Arg Leu Ser Pro 195 200 205Ser Gln Ser Pro Ile Asp His Ser Leu Ser Asp Thr Leu 210 215 220 5 1642DNA Arabidopsis thaliana 5 tagaagtggt agtggagtga aaaaacaaat cctaagcagtcctaaccgat ccccgaagct 60 aaagattctt caccttccca aataaagcaa aacctagatccgacattgaa ggaaaaacct 120 tttagatcca tctctgaaaa aaaaccaacc atgaagagagatcatcatca tcatcatcaa 180 gataagaaga ctatgatgat gaatgaagaa gacgacggtaacggcatgga tgttgctcag 240 aaactcgagc agcttgaagt tatgatgtct aatgttcaagaagacgatct ttctcaactc 300 gctactgaga ctgttcacta taatccggcg gagctttacacgtggcttga ttctatgctc 360 accgacctta atcctccgtc gtctaacgcc gagtacgatcttaaagctat tcccggtgac 420 gcgattctca atcagttcgc tatcgattcg gcttcttcgtctaaccaagg cggcggagga 480 gatacgtata ctacaaacaa gcggttgaaa tgctcaaacggcgtcgtgga aaccaccaca 540 gcgacggctg agtcaactcg gcatgttgtc ctggttgactcgcaggagaa cggtgtgcgt 600 ctcgttcacg cgcttttggc ttgcgctgaa gctgttcagaaggagaatct gactgtggcg 660 gaagctctgg tgaagcaaat cggattctta gctgtttctcaaatcggagc tatgagaaaa 720 gtcgctactt acttcgccga agctctcgcg cggcggatttaccgtctctc tccgtcgcag 780 agtccaatcg accactctct ctccgatact cttcagatgcacttctacga gacttgtcct 840 tatctcaagt tcgctcactt cacggcgaat caagcgattctcgaagcttt tcaagggaag 900 aaaagagttc atgtcattga ttctctatga gtcaaggtcttcaatggccg gcgcttatgc 960 aggctcttgc gcttcgacct ggtggtcctc ctgttttccggttaaccgga attggtccac 1020 cggcaccgga taatttcgat tatcttcatg aagttgggtgtaagctggct catttagctg 1080 aggcgattca cgttgagttt gagtacagag gatttgtggctaacacttta gctgatcttg 1140 atgcttcgat gcttgagctt agaccaagtg agattgaatctgttgcggtt aactctgttt 1200 tcgagcttca caagctcttg ggacgacctg gtgcgatcgataaggttctt ggtgtggtga 1260 atcagattaa accggagatt ttcactgtgg ttgagcaggaatcgaaccat aatagtccga 1320 ttttcttaga tcggtttact gagtcgttgc attattactcgacgttgttt gactcgttgg 1380 aaggtgtacc gagtggtcaa gacaaggtca tgtcggaggtttacttgggt aaacagatct 1440 gcaacgttgt ggcttgtgat ggacctgacc gagttgagcgtcatgaaacg ttgagtcagt 1500 ggaggaaccg gttcgggtct gctgggtttg cggctgcacatattggttcg aatgcgttta 1560 agcaagcgag tatgcttttg gctctgttca acggcggtgagggttatcgg gtggaggaga 1620 gtgacggctg tctcatgttg gg 1642 6 259 PRTArabidopsis thaliana 6 Met Lys Arg Asp His His His His His Gln Asp LysLys Thr Met Met 1 5 10 15 Met Asn Glu Glu Asp Asp Gly Asn Gly Met AspVal Ala Gln Lys Leu 20 25 30 Glu Gln Leu Glu Val Met Met Ser Asn Val GlnGlu Asp Asp Leu Ser 35 40 45 Gln Leu Ala Thr Glu Thr Val His Tyr Asn ProAla Glu Leu Tyr Thr 50 55 60 Trp Leu Asp Ser Met Leu Thr Asp Leu Asn ProPro Ser Ser Asn Ala 65 70 75 80 Glu Tyr Asp Leu Lys Ala Ile Pro Gly AspAla Ile Leu Asn Gln Phe 85 90 95 Ala Ile Asp Ser Ala Ser Ser Ser Asn GlnGly Gly Gly Gly Asp Thr 100 105 110 Tyr Thr Thr Asn Lys Arg Leu Lys CysSer Asn Gly Val Val Glu Thr 115 120 125 Thr Thr Ala Thr Ala Glu Ser ThrArg His Val Val Leu Val Asp Ser 130 135 140 Gln Glu Asn Gly Val Arg LeuVal His Ala Leu Leu Ala Cys Ala Glu 145 150 155 160 Ala Val Gln Lys GluAsn Leu Thr Val Ala Glu Ala Leu Val Lys Gln 165 170 175 Ile Gly Phe LeuAla Val Ser Gln Ile Gly Ala Met Arg Lys Val Ala 180 185 190 Thr Tyr PheAla Glu Ala Leu Ala Arg Arg Ile Tyr Arg Leu Ser Pro 195 200 205 Ser GlnSer Pro Ile Asp His Ser Leu Ser Asp Thr Leu Gln Met His 210 215 220 PheTyr Glu Thr Cys Pro Tyr Leu Lys Phe Ala His Phe Thr Ala Asn 225 230 235240 Gln Ala Ile Leu Glu Ala Phe Gln Gly Lys Lys Arg Val His Val Ile 245250 255 Asp Ser Leu 7 1636 DNA Arabidopsis thaliana 7 tagaagtggtagtggagtga aaaaacaaat cctaagcagt cctaaccgat ccccgaagct 60 aaagattcttcaccttccca aataaagcaa aacctagatc cgacattgaa ggaaaaacct 120 tttagatccatctctgaaaa aaaaccaacc atgaagagag atcatcatca tcatcatcaa 180 gataagaagactatgatgat gaatgaagaa gacgacggta acggcatgga tgttgctcag 240 aaactcgagcagcttgaagt tatgatgtct aatgttcaag aagacgatct ttctcaactc 300 gctactgagactgttcacta taatccggcg gagctttaca cgtggcttga ttctatgctc 360 accgaccttaatcctccgtc gtctaacgcc gagtacgatc ttaaagctat tcccggtgac 420 gcgattctcaatcagttcgc tatcgattcg gcttcttcgt ctaaccaagg cggcggagga 480 gatacgtatactacaaacaa gcggttgaaa tgctcaaacg gcgtcgtgga aaccaccaca 540 gcgacggctgagtcaactcg gcatgttgtc ctggttgact cgcaggagaa cggtgtgcgt 600 ctcgttcacgcgcttttggc ttgcgctgaa gctgttcaga aggagaatct gactgtggcg 660 gaagctctggtgaagcaaat cggattctta gctgtttctc aaatcggagc tatgagaaaa 720 gtcgctacttacttcgccga agctctcgcg cggcggattt accgtctctc tccgtcgcag 780 agtccaatcgaccactctct ctccgatact cttcagatgc acttctacga gacttgtcct 840 tatctcaagttcgctcactt cacggcgaat caagcgattc tcgaagcttt tcaagggaag 900 aaaagagttcatgtcattga tttctctatg agtcaaggtc ttgggcgctt atgcaggctc 960 ttgcgcttcgacctggtggt cctcctgttt tccggttaac cggaattggt ccaccggcac 1020 cggataatttcgattatctt catgaagttg ggtgtaagct ggctcattta gctgaggcga 1080 ttcacgttgagtttgagtac agaggatttg tggctaacac tttagctgat cttgatgctt 1140 cgatgcttgagcttagacca agtgagattg aatctgttgc ggttaactct gttttcgagc 1200 ttcacaagctcttgggacga cctggtgcga tcgataaggt tcttggtgtg gtgaatcaga 1260 ttaaaccggagattttcact gtggttgagc aggaatcgaa ccataatagt ccgattttct 1320 tagatcggtttactgagtcg ttgcattatt actcgacgtt gtttgactcg ttggaaggtg 1380 taccgagtggtcaagacaag gtcatgtcgg aggtttactt gggtaaacag atctgcaacg 1440 ttgtggcttgtgatggacct gaccgagttg agcgtcatga aacgttgagt cagtggagga 1500 accggttcgggtctgctggg tttgcggctg cacatattgg ttcgaatgcg tttaagcaag 1560 cgagtatgcttttggctctg ttcaacggcg gtgagggtta tcgggtggag gagagtgacg 1620 gctgtctcatgttggg 1636 8 282 PRT Arabidopsis thaliana 8 Met Lys Arg Asp His His HisHis His Gln Asp Lys Lys Thr Met Met 1 5 10 15 Met Asn Glu Glu Asp AspGly Asn Gly Met Asp Val Ala Gln Lys Leu 20 25 30 Glu Gln Leu Glu Val MetMet Ser Asn Val Gln Glu Asp Asp Leu Ser 35 40 45 Gln Leu Ala Thr Glu ThrVal His Tyr Asn Pro Ala Glu Leu Tyr Thr 50 55 60 Trp Leu Asp Ser Met LeuThr Asp Leu Asn Pro Pro Ser Ser Asn Ala 65 70 75 80 Glu Tyr Asp Leu LysAla Ile Pro Gly Asp Ala Ile Leu Asn Gln Phe 85 90 95 Ala Ile Asp Ser AlaSer Ser Ser Asn Gln Gly Gly Gly Gly Asp Thr 100 105 110 Tyr Thr Thr AsnLys Arg Leu Lys Cys Ser Asn Gly Val Val Glu Thr 115 120 125 Thr Thr AlaThr Ala Glu Ser Thr Arg His Val Val Leu Val Asp Ser 130 135 140 Gln GluAsn Gly Val Arg Leu Val His Ala Leu Leu Ala Cys Ala Glu 145 150 155 160Ala Val Gln Lys Glu Asn Leu Thr Val Ala Glu Ala Leu Val Lys Gln 165 170175 Ile Gly Phe Leu Ala Val Ser Gln Ile Gly Ala Met Arg Lys Val Ala 180185 190 Thr Tyr Phe Ala Glu Ala Leu Ala Arg Arg Ile Tyr Arg Leu Ser Pro195 200 205 Ser Gln Ser Pro Ile Asp His Ser Leu Ser Asp Thr Leu Gln MetHis 210 215 220 Phe Tyr Glu Thr Cys Pro Tyr Leu Lys Phe Ala His Phe ThrAla Asn 225 230 235 240 Gln Ala Ile Leu Glu Ala Phe Gln Gly Lys Lys ArgVal His Val Ile 245 250 255 Asp Phe Ser Met Ser Gln Gly Leu Gly Arg LeuCys Arg Leu Leu Arg 260 265 270 Phe Asp Leu Val Val Leu Leu Phe Ser Gly275 280 9 1642 DNA Arabidopsis thaliana 9 tagaagtggt agtggagtgaaaaaacaaat cctaagcagt cctaaccgat ccccgaagct 60 aaagattctt caccttcccaaataaagcaa aacctagatc cgacattgaa ggaaaaacct 120 tttagatcca tctctgaaaaaaaaccaacc atgaagagag atcatcatca tcatcatcaa 180 gataagaaga ctatgatgatgaatgaagaa gacgacggta acggcatgga tgttgctcag 240 aaactcgagc agcttgaagttatgatgtct aatgttcaag aagacgatct ttctcaactc 300 gctactgaga ctgttcactataatccggcg gagctttaca cgtggcttga ttctatgctc 360 accgacctta atcctccgtcgtctaacgcc gagtacgatc ttaaagctat tcccggtgac 420 gcgattctca atcagttcgctatcgattcg gcttcttcgt ctaaccaagg cggcggagga 480 gatacgtata ctacaaacaagcggttgaaa tgctcaaacg gcgtcgtgga aaccaccaca 540 gcgacggctg agtcaactcggcatgtgtcc tggttgactc gcaggagaac ggtgtgcgtc 600 tcgttcacgc gcttttggcttgcgctgaag ctgttcagaa ggagaatctg actgtggcgg 660 aagctctggt gaagcaaatcggattcttag ctgtttctca aatcggagct atgagaaaag 720 tcgctactta cttcgccgaagctctcgcgc ggcggattta ccgtctctct ccgtcgcaga 780 gtccaatcga ccactctctctccgatactc ttcagatgca cttctacgag acttgtcctt 840 atctcaagtt cgctcacttcacggcgaatc aagcgattct cgaagctttt caagggaaga 900 aaagagttca tgtcattgatttctctatga gtcaaggtct tcaatggccg gcgcttatgc 960 aggctcttgc gcttcgacctggtggtcctc ctgttttccg gttaaccgga attggtccac 1020 cggcaccgga taatttcgattatcttcatg aagttgggtg taagctggct catttagctg 1080 aggcgattca cgttgagtttgagtacagag gatttgtggc taacacttta gctgatcttg 1140 atgcttcgat gcttgagcttagaccaagtg agattgaatc tgttgcggtt aactctgttt 1200 tcgagcttca caagctcttgggacgacctg gtgcgatcga taaggttctt ggtgtggtga 1260 atcagattaa accggagattttcactgtgg ttgagcagga atcgaaccat aatagtccga 1320 ttttcttaga tcggtttactgagtcgttgc attattactc gacgttgttt gactcgttgg 1380 aaggtgtacc gagtggtcaagacaaggtca tgtcggaggt ttacttgggt aaacagatct 1440 gcaacgttgt ggcttgtgatggacctgacc gagttgagcg tcatgaaacg ttgagtcagt 1500 ggaggaaccg gttcgggtctgctgggtttg cggctgcaca tattggttcg aatgcgttta 1560 agcaagcgag tatgcttttggctctgttca acggcggtga gggttatcgg gtggaggaga 1620 gtgacggctg tctcatgttggg 1642 10 166 PRT Arabidopsis thaliana 10 Met Lys Arg Asp His His HisHis His Gln Asp Lys Lys Thr Met Met 1 5 10 15 Met Asn Glu Glu Asp AspGly Asn Gly Met Asp Val Ala Gln Lys Leu 20 25 30 Glu Gln Leu Glu Val MetMet Ser Asn Val Gln Glu Asp Asp Leu Ser 35 40 45 Gln Leu Ala Thr Glu ThrVal His Tyr Asn Pro Ala Glu Leu Tyr Thr 50 55 60 Trp Leu Asp Ser Met LeuThr Asp Leu Asn Pro Pro Ser Ser Asn Ala 65 70 75 80 Glu Tyr Asp Leu LysAla Ile Pro Gly Asp Ala Ile Leu Asn Gln Phe 85 90 95 Ala Ile Asp Ser AlaSer Ser Ser Asn Gln Gly Gly Gly Gly Asp Thr 100 105 110 Tyr Thr Thr AsnLys Arg Leu Lys Cys Ser Asn Gly Val Val Glu Thr 115 120 125 Thr Thr AlaThr Ala Glu Ser Thr Arg His Val Ser Trp Leu Thr Arg 130 135 140 Arg ArgThr Val Cys Val Ser Phe Thr Arg Phe Trp Leu Ala Leu Lys 145 150 155 160Leu Phe Arg Arg Arg Ile 165 11 15 DNA Arabidopsis thaliana 11 tagaagtggtagtgg 15 12 16 DNA Arabidopsis thaliana 12 accatgagac cagccg 16

What is claimed is:
 1. A nucleic acid isolate having a nucleotidesequence coding for a polypeptide which comprises the amino acidsequence shown in FIG. 4 (SEQ ID NO:2).
 2. Nucleic acid according toclaim 1 wherein the coding nucleotide sequence comprises the codingnucleotide sequence shown in FIG. 3 (SEQ ID NO:1).
 3. Nucleic acidaccording to claim 1 wherein the coding nucleotide sequence encodes theamino acid sequence shown in FIG. 4 (SEQ ID NO:2) but differs from thecoding nucleotide sequence shown in FIG. 3 (SEQ ID NO:1).
 4. Nucleicacid according to claim 1 further including a regulatory sequence forexpression from said coding nucleotide sequence.
 5. Nucleic acidaccording to claim 4 wherein the regulatory sequence includes aninducible promoter.
 6. A nucleic acid vector suitable for transformationof a plant cell and including nucleic acid according to claim
 1. 7. Ahost cell containing the nucleic acid according to claim 1, wherein saidnucleic acid is heterologous to said host cell.
 8. A host cell accordingto claim 7 which is microbial.
 9. A host cell according to claim 7 whichis a plant cell.
 10. A plant cell according to claim 9 having saidheterologous nucleic acid within its genome.
 11. A plant cell accordingto claim 9 which is comprised in a plant, a plant part or a plantpropagule, or derivative of a plant.
 12. A plant comprising a plant cellaccording to claim
 9. 13. A part or propagule, or derivative of a plant,comprising a plant cell according to claim
 9. 14. A plant cell accordingto claim 10 having more than one said nucleotide sequence per haploidgenome.
 15. A method of producing a cell according to claim 7, themethod including incorporating said nucleic acid into the cell by meansof transformation.
 16. A method according to claim 15 which includesrecombining the nucleic acid with the cell genome nucleic acid such thatit is stably incorporated therein.
 17. A method according to claim 15which includes regenerating a plant from one or more transformed cells.18. A method of producing a plant, the method including incorporatingnucleic acid according to claim 1 into a plant cell and regenerating aplant from said plant cell.
 19. A method according to claim 18 includingsexually or asexually propagating or growing off-spring or a descendantof the plant regenerated from said plant cell.
 20. A method ofinfluencing a characteristic of a plant, which characteristic isselected from plant growth and delayed flowering in short dayconditions, the method including causing or allowing expression fromheterologous nucleic acid according to claim 1 within cells of theplant.